This invention relates to sterilization processes and more specifically to sterilization processes which are particularly suited for biological materials, such as organ replacements, and which methods exhibit efficacy against difficult-to-kill bacteria and bacterial spores.
Sterilization techniques are widely used and important in industries such as food processing and health care. Saturated steam at temperatures above 110xc2x0 C. has frequently been used to destroy microorganisms, such as microbial spores. Certain articles, particularly those used for health care, cannot withstand the temperatures and moisture of steam sterilization, and oftentimes such articles are also considered not to be suitable for sterilization by ionizing radiation. As a result, gaseous sterilants have been developed which function at relatively low temperatures and thus offer an attractive alternative. One of the most commonly used gaseous sterilants is ethylene oxide, which is used for medical product sterilization and for other sterilization processes. However, in certain instances, the presence of residual ethylene oxide, even in small quantities, is considered to be detrimental, and accordingly improved sterilization processes, particularly for sterilization of medical products, have continued to be sought.
It has now been found that sterilization of items, including biological tissue, replacement organs and synthetic prosthetic materials, including polymers and metals, can be effectively carried out by treatment with a coupling agent, e.g. a water-soluble carbodiimide, that is capable of creating amide linkages between amines and carboxylic acids; such treatment has been proven to be bactericidal. Sterilization treatment is carried out at a temperature above ambient, and although it may employ an optional coupling enhancer, such is not felt necessary. Treatment may be carried out using an organic solution of an appropriate coupling agent or using an aqueous buffered solution that may optionally contain isopropyl alcohol or the like, but the presence of such an alcohol is not necessary to achieve effective sterilization. The residuals from such treatment are nontoxic, biocompatible, and water-soluble; they can generally be easily be washed off the tissue before implantation in a human body. Surprisingly, biological tissue which has been effectively sterilized using a water-soluble carbodiimide may exhibit enhanced resistance to degeneration and/or calcification following its implantation within a living body.
The term xe2x80x9ccoupling agentxe2x80x9d is herein used to refer to a chemical reagent that facilitates the formation of amide bonds. Such bonds may be formed between reactive amines and reactive carboxyls on enzymes and proteins as well as between the reactive carboxyl or amine moieties located on and within bioprosthetic tissue. Those having skill in peptide synthesis and related arts will be familiar with some such reagents, e.g. water-soluble carbodiimides and succinimides; there are other known coupling agents that are soluble in organic solvents. When penetration of such coupling agents into the cells of microorganisms occurs, it results in sterilization, destroying bacteria, spores and possibly viruses and other infectious agents by internal cross-linking. When biological tissue is to be treated, the coupling agent chosen is preferably one that is water-soluble so the treatment can be effected in aqueous solution at a physiological pH. When other materials that are resistant to organic solvents are to be sterilized, e.g. synthetic polymeric materials, organic solutions of appropriately soluble coupling agents may be used. Any suitable carbodiimide can be used as the coupling agent; however, the preferred water-soluble coupling agent is 1-ethyl-3(3-dimethyl aminopropyl)carbodiimide hydro-chloride (EDC). Other water-soluble carbodiimides include 1-Cyclohexyl-3(2-morpholinoethyl)carbodiimide, N,Nxe2x80x2-Carbonyldiimidazole, Woodward""s Reagent K, and mixtures of such carbodiimides. A list of such cross-linking coupling agents can be found in the book: Bioconjugate Techniques by Greg T. Hermanson published by Academic Press 1996, the relevant disclosure of which is incorporated herein by reference. When biological tissue is being treated, the water-soluble coupling agent EDC is preferably used. As indicated above, an optional enhancer, e.g. N-hydroxysulfosuccinimide (sulfo-NHS), might be included at a concentration between 0.5 mM and about 30 mM when EDC is used as the coupling agent; however, such is not considered necessary for effective sterilization.
The sterilization treatment is considered to be temperature-dependent, with a relatively low temperature of about 35-40xc2x0 C. being preferred because of its lack of potential adverse effect upon the material being treated. The concentration of the coupling agent can be varied within a reasonable range, and treatment with higher concentrations of the coupling agent has been found to achieve sterilization within a shorter time of treatment. Although lower concentrations, e.g. 5-15 mM, may be effectively used, particularly when higher temperatures are employed, the coupling agent is preferably used in a concentration between about 25 millimolar (mM) and about 150 mM, more preferably between about 35 mM and about 100 mM, and most preferably at between about 50 mM and about 75 mM, in order to be certain of destroying all commonly encountered bacteria and spores within a reasonable duration of treatment.
Higher concentrations of coupling agent, so long as compatible with the material being sterilized, will generally reduce the duration of treatment needed. It has been found that effective sterilization is achieved when such treatment is carried out at a suitable temperature above ambient, e.g. at a temperature of at least about 35xc2x0 C., for a minimum number of concentration-duration units, i.e. a multiple of coupling agent concentration and duration of exposure. By arbitrarily basing such units upon millimoles of the coupling agent and hours of sterilization treatment, it has been found that at a temperature of about 35-40xc2x0 C., a minimum number of units equal to at least about 450 millimole hours should be employed. For example, effective sterilization may be achieved at a coupling agent concentration of about 50 mM for about 9-10 hours or alternatively at a concentration of about 25 mM for about 20-24 hours. At a concentration of about 120 mM, treatment at about 40xc2x0 C. for about 6 hours should achieve sterilization. Although even higher concentrations, e.g. 150 mM, might be used, they are considered to be generally unnecessary and likely impractical from an economic standpoint. Moreover, by raising the temperature, e.g. to about 50-55xc2x0 C., treatment for at least about 100-150 millimole hours should suffice, e.g. 3 hours with a concentration of 50 mM or 10 hours at 10 mM. Obviously, longer durations can be employed, and for purposes of safety, it may be desirable to employ such sterilization treatment for about 25-50% longer than the above-stated minimum that should achieve sterilization under normal conditions.
For purposes of this application, a particular sterilization treatment is deemed to be effective when it will effect a reduction of about 106 (6 log) when about 106 spores and/or microorganisms are inoculated in the test sample. This should assure that there will be no survivors in actual practice because biological tissue or other material that is being subjected to a sterilization treatment will not reasonably contain a level of microorganism contamination even approaching this magnitude. This treatment not only achieves sterilization without risk of damage to biological tissue that is to be implanted, but it may also make some contribution to stability of certain fixed biological tissue, e.g. the resistance of such biological tissue to degenerate and/or calcify within a living body may be enhanced.
Reaction conditions for the sterilization treatment may vary somewhat depending on the specific coupling agent employed. Sterilization treatment is frequently carried out using a water-soluble carbodiimide in an aqueous buffer solution selected from among buffers that are well known to those of ordinary skill in this art as being suitable buffers for use in the physiological pH range. Examples of suitable buffers include, but are not limited to, N-2-hydroxyethylpiperazine-Nxe2x80x2-ethanesulfonic acid (HEPES), Tris(hydroxymethyl)aminomethane, 3-(N-morpholino)propanesulfonic acid (MOPS), N-Tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid, N-Tris(hydroxymethyl)methylglycine, and the like.
The pH and concentration of buffer in an aqueous solution also may vary depending upon the coupling agent employed. The buffer concentration and pH are chosen to provide an effective sterilization environment while being the least harmful to bioprosthetic or other material being treated. For example, with EDC as the coupling agent for sterilizing biological tissue, the pH of the aqueous solution employed is about 6.0 to about 7.0 and the temperature is usually maintained between about 35xc2x0 C. and 40xc2x0 C. As mentioned above, higher temperatures may be used so long as they are compatible with the materials being sterilized; however, temperatures above about 55xc2x0 C. are not generally used, for example, with medical devices made of bioprosthetic tissue. Preferably, sterilization is carried out at about 40xc2x0 C. or above. For sterilizing polymeric or metallic materials, temperatures slightly higher than 55xc2x0 C., i.e. about 75xc2x0 C., may be used so long as they are not harmful to the material being sterilized, and use of such higher temperatures may shorten the necessary duration of treatment. All sterilization treatment solutions are preferably filtered through 0.45 xcexcm or smaller filters before use to eliminate possible contamination. The optional inclusion in such solution of a small volume % of a C2 to C4 alkanol or an equivalent alcohol may permit sterilization at a lower temperature and/or with a lower percentage of coupling agent; however, isopropanol or the like may have an adverse effect upon certain tissue, in which case such sterilization should be carried out in the absence of an alkanol.
This sterilization treatment method is considered useful for a wide variety of prosthetic and bioprosthetic materials; however, it is considered to be particularly useful for sterilizing replacement organ components, such as heart valves, which have been made from animal tissue that has been suitably fixed. By suitably fixed is meant having been subjected to cross-linking (xe2x80x9cfixingxe2x80x9d), as by glutaraldehyde treatment or by a comparable process such as described in U.S. Pat. Nos. 5,447,536 and 5,733,339 so as to raise the shrinkage temperature thereof. Other fixation techniques, e.g. polyepoxide crosslinking or photo-oxidation may also be used. In some instances, it may be important that, if biological tissue is being fixed, it has been at least minimally fixed; otherwise, the chemical action of the coupling agent might be diverted.
It may be desirable to first rinse the material with cold saline prior to sterilization as a preparation therefor, because sterilization is usually a final step before use or packaging. The material being sterilized is usually maintained in contact with the sterilization solution for about 6 to 48 hours, and such treatment has been shown to effectively inactivate even hard-to-kill bacteria and spores, thus proving the process to be potently bactericidal. It is common in the industry to xe2x80x9cquarantinexe2x80x9d such sterilized products for 1 to 2 weeks, during which time the bioindicator results of sterilization will be received. The product is then considered ready for packaging or temporary storage. Thereafter, it is ready for immediate use following one or more sterile rinses to remove any remaining unreacted reagents and by-products. This sterilization treatment has not been shown to adversely affect bioprosthetic tissue, as by possibly lowering the shrinkage temperature of such sterilized material or by lowering its resistance to proteolytic degradation by collagenase or by proteases; on the contrary, in some instances, it may increase shrinkage temperature and/or surprisingly increase resistance of biological tissue to calcification.
The present invention is further described by the examples that follow. These examples are not to be construed as limiting in any way either the spirit or the scope of the present invention.
Devices to be implanted in the human body are required to be sterilized in a manner to effectively destroy all microorganisms. Due to the unique applications of liquid chemicals for use in sterilization processes, it is necessary to be vigilant in detecting, screening and testing microorganisms which could pose significant resistance to the sterilization process. Examples of reference microorganisms which have previously demonstrated high resistance to liquid chemical sterilants are: the spores of Bacillus subtilis, Clostridium sporogenes, Bacillus pumilus, Chaetonium globosom and Microascus cinereus and representative vegetative cells, such as Mycobacterium chelonae, Methylbactrium extorquens, and Trichosporon aquatile. Of the foregoing, the most resistant may be the spores of Bacillus subtilis. The coupling agent used in the following examples is 1-ethyl-3(3-dimethyl aminopropyl)carbodiimide hydrochloride (EDC), and when used, the optional enhancer is either N-hydroxysulfosuccinimide (sulfo-NHS) or hydroxysuccinimide (NHS), which are all commercially available. Peptone water is prepared by dissolving 1 g of Bacto Peptone in 1 liter of de-ionized water. The solution is then filtered into sterile bottles using sterile 0.2 micron filters. All agents are solubilized in 10 mM HEPES buffer containing 0.85% of sodium chloride, pH 6.5 (HEPES buffer). Concentrations are expressed as mM (number of millimoles of chemical for each liter of solution), or as % (grams per 100 ml of solution). Temperatures are in xc2x0 C. (degrees Celsius), with room temperature being about 20-25xc2x0.
Porcine aortic roots are fixed by cross-linking according to the method described in U.S. Pat. No. 5,447,536. After fixation, the valves are stored in 10 mM HEPES, 0.85% NaCl, 20% isopropyl alcohol, pH 7.4, at 4xc2x0 C. The sterility tests described in the following examples are in most cases conducted in the presence of bioprosthetic heart valve tissue; when such tissue is not present, the solutions are simply filtered through a 0.45 micron filter attached to a funnel (filter funnel). The filters are then rinsed with peptone water to eliminate residual chemicals on the membrane that may prevent growth of the organisms tested. Such membrane filters are then incubated on TSA plates at about 32xc2x0 to 33xc2x0 C., e.g. 32.5xc2x0 C. When an aortic valve is inoculated with microorganisms for test purposes and then submitted to sterilization, the solution is filtered as described above. The aortic valve tissue is then washed for 20 minutes in a reciprocating shaker in the presence of peptone water containing Tween 80 in order to extract all indigenous spores or microorganisms from the tissue. This solution is filtered and then incubated as described above. All microbiological testing is performed in a biological laminar flow hood to prevent contamination. The shrinkage temperature and the proteolytic (collagenase and protease) degradation tests are conducted as previously described in the ""536 patent. Resistance to calcification is assessed by subdermal implantation of sterilized leaflets and aortic wall coupons in young rats, as also described in the ""536 U.S. patent.